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(Chest. 2003;124:306-313.)
© 2003 American College of Chest Physicians

Hyperosmolar Solutions Stimulate Mucus Secretion in the Ferret Trachea*

Chikako Kishioka, MD, PhD; Kosuke Okamoto, MD, PhD; Jung-Soo Kim, MD, PhD and Bruce K. Rubin, MD, FCCP

* From the Department of Otolaryngology (Drs. Kishioka and Okamoto), Mie University School of Medicine, Tsu, Mie, Japan; and Wake Forest University School of Medicine (Drs. Kim and Rubin), Winston-Salem, NC.

Correspondence to: Bruce K. Rubin, MD, FCCP, Professor and Vice-Chair, Department of Pediatrics, Professor of Medical Engineering, Medicine, Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1081; e-mail: brubin{at}wfubmc.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Inhalation of hypertonic saline or mannitol solutions acutely increases mucociliary clearance. Because increased clearance is often coupled with increased mucus secretion, we hypothesized that hyperosmolar agents would stimulate mucus secretion.

Setting and subjects: The isolated tracheae of healthy young adult ferrets were studied in a basic research laboratory.

Measurements and results: We demonstrated that there was a dose-dependent increase in mucin secretion by enzyme-linked lectin assay after incubation with 1.69 g/dL (597 mOsm/L), 3.69 g/dL (1,192 mOsm/L), 5.69 g/dL (1,823 mOsm/L), and 10.69 g/dL (3,612 mOsm/L) of saline solution over Krebs-Henseleit solution control (288 mOsm/L) [p < 0.01 for 1.69 g/dL of saline solution and p < 0.0001 for others]. Mannitol solution, 15 g/dL (1,040 mOsm/L), also significantly increased mucin secretion (n = 4, p < 0.005). There was a 47% and 54% increase in secretion of the serous cell product lysozyme after exposure to 3.69 g/dL (1,192 mOsm/L) and 10.69 g/dL (3,612 mOsm/L) saline solutions, respectively (n = 5, p < 0.05). Secretion was only stimulated when the hyperosmolar exposure was on the luminal side of the epithelium. Mucin secretion was induced within minutes of 3.69 g/dL of saline solution exposure, and this increased mucin secretion quickly peaked. The ratio of mucin to lysozyme secretion was approximately 2. This ratio appeared to be independent of the osmotic concentration of the stimulus and therefore of secretory rate.

Conclusions: Mucus secretion is markedly stimulated in response to hyperosmolarity. This may be a protective response. These results also suggest that the therapeutic use of hyperosmolar aerosols should be evaluated with care when used for patients with mucus hypersecretion and impaired mucus clearance.

Key Words: hypertonic saline solution • inhalation administration • mucus • mucociliary clearance


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sputum induction using hypertonic saline solution inhalation has been used to obtain sputum specimens for diagnosing airway infection1 and for collecting secretions for research.2 3 More than 20 years ago, it was shown that when patients with chronic bronchitis inhaled a 7% (7 g/dL, 2,396 mOsm/L) saline aerosol, whole-lung mucociliary clearance (MCC) increased by 100% in the first 90 min over the clearance rate after inhalation of isotonic (0.9%) saline solution.4 More recently, patients with cystic fibrosis CF) were randomly assigned to receive either 0.9% saline or 6% saline by ultrasonic nebulization.5 6 Spirometry was measured before initiating the study, at day 14 after 2 weeks of study drug, and at day 28, 2 weeks after therapy was discontinued. Among the patients who completed these studies, there was a significant increase in FEV1 in those inhaling hypertonic saline solution, and this returned to baseline by day 28.

The mechanism by which hyperosmolar agents increase MCC has not been established. The increase in MCC could be due to an increase in airway secretion volume,7 an increase in ciliary activity,8 or a change in the properties of the secretions.8 9 We hypothesized that hyperosmolar solutions would increase mucin (mucous cell) and lysozyme (serous cell) secretion in the ferret trachea in a dose-dependent manner.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Airway mucus is composed of secretory products produced from different cell populations. In the larger airways of many mammals, including man, the submucosal glands and epithelial goblet cells are the major sources of mucus. The ferret trachea is a robust model to study airway mucus secretion because of its long length and high glandularity similar to the human airway and unlike other small animals.10 We have studied the regulation of mucous and serous cell secretion under a variety of experimental conditions using the ferret tracheal model.11 12 13

Mucin and Lysozyme Secretion
Adult ferrets (1.7 to 2.3 kg, random sex) obtained from Marshall Farms (North Rose, NY) were killed by intraperitoneal injection of pentobarbital sodium (150 mg/kg body weight), and the trachea from the larynx to the carina was immediately removed. Each trachea was divided into four or eight roughly equal segments from the upper trachea to the carina. The segments were weighed and then immersed in 10 mL of Krebs-Henseleit solution (KHS) [containing 6.9 g/L of NaCl, 0.35 g/L of KCl, 0.16 g/L of KH2PO4, 0.141 g/L of MgSO4, 0.373 g/L of CaCl2, 2 g/L of D-glucose: pH 7.4, 255 mOsm/L]. This was agitated gently in a shaking water bath at ferret body temperature, 38°C, with 0.5 L/min of 100% oxygen provided into the incubator bath. Measured fraction of inspired oxygen varied from 0.40 to 0.45. Isolated tracheae were rested in KHS for 2 h to stabilize airway secretion. The incubation solution was changed, and the segments were incubated for 20 min with only KHS to measure the constitutive secretion (period 1). These were then incubated for another 20 min with test agent: KHS with added 1 g/dL of saline solution (597 mOsm), 3 g/dL of saline solution (1,192 mOsm), 5 g/dL of saline solution (1,823 mOsm), 10 g/dL of saline solution (3,612 mOsm), or 15 g/dL of mannitol solution (1,040 mOsm), or with only KHS (0.69 g/dL of saline solution [288 mOsm]) as a negative control (period 2). After the experiment, the bathing solution was collected and stored at - 70°C until analyzed.

The relative contribution of mucous and serous cells to the secretion was evaluated by measuring the amount of mucin, a marker for mucous cell secretion,11 12 13 and lysozyme as a marker of serous cell secretion.14 A secretory index (SI) expressing the relative increase in mucin or lysozyme under experimental conditions was calculated for each tracheal segment as concentration after a 20-min exposure to the test agent (period 2) divided by the concentration measured after a 20-min exposure to KHS alone (period 1). We randomized the administration of agents to different segments in each experiment. There is anatomic variation in the amount of ferret tracheal mucin secretion, with significantly greater secretion on the proximal (cephalad) trachea than near the carina.11 We eliminated anatomic variation in secretion as a potential confounding variable by ensuring that each study group had an equal number of segments from each portion of the trachea. Each tracheal segment was exposed to only one test agent.

The concentrations of saline solution used were based on published data demonstrating that the hypertonic saline solution increases mucociliary clearance at a concentration of 3 to 12%.3 4 5 15 The concentration of 14 g/dL of mannitol was chosen because it has an osmolarity similar to that of 3.69 g/dL of saline solution (1,040 mOsm vs 1,192 mOsm, respectively). The osmolarity of all solution was measured by freezing-point depression in the Clinical Laboratory Improvement Amendments-certified clinical laboratory of our hospital. Measurements were performed in triplicate and agreed within 0.5%. These results were also consistent with the calculated osmolarity of saline solutions. These studies were approved by the Animal Care and Use Committee of Wake Forest University.

Mucin Secretion by Enzyme-Linked Lectin Assay
Submucosal glands in the ferret trachea have both serous and mucous cells, and ferret tracheal mucins have high blood group titers, reflecting an abundance of galactose-N-acetyl-{alpha}1–3 (fucose-{alpha}1–2) galactose-R.16 These blood group antigens can be detected by the lectin Dolichos biflorus agglutinin (DBA). DBA immunohistochemistry staining shows specific binding to goblet cells and submucosal glands in the ferret trachea.12

A sandwich enzyme-linked lectin assay was used to measure mucin secretion as previously described.11 In brief, a 96-well microtiter plate was coated with 60 µL of DBA (6 µg/mL in phosphate-buffered saline solution [PBS]) and incubated at room temperature for 2 h. After rinsing with high-salt PBS (PBS containing 0.5 mol/L NaCl and 0.1% Tween 20), the plate was exposed to sample buffer and incubated at room temperature for 1 h. This was then treated with 50 µL of DBA conjugated with horseradish peroxidase (0.25 µg/mL) in PBS containing 1% bovine serum albumin. Before and after this setup, this plate was washed with high-salt PBS. Tetramethylbenzidine, 150 µL (0.42 mmol/L), in citrate-acetate buffer (pH 6.0) was added to each well and incubated for 10 min. The reaction was stopped by adding 50 µL of H2SO4 (normal, 4.7), Color development was read as the difference in adsorbance at 450 nm and 650 nm. The concentration of mucin was calculated by comparison with standard mucin (asialo ovine submaxillary mucin: 20 to 200 ng/mL). The amount of mucin secreted was expressed mucin weight per trachea tissue weight (nanograms per gram) Tissue weight was used as a more accurately measured surrogate for secretory surface area.

Lysozyme Secretion by Spectrophotometry
Lysozyme in a bacteriolytic enzyme. It has been reported that its only source in the ferret airway is the serous cells of submucosal glands.14 Lysozyme activity was determined spectrophotometrically by measuring the initial rate of lysis of a 1.38 mg/mL Micrococcus lysodeikticus suspension. A 0.1-mL volume of sample buffer was added to 1.4 mL of substrate in PBS buffer at pH 6.0. The change in turbidity was measured at 540 nm. One unit of enzyme activity was defined as the change in adsorbance per minute equivalent to that produced by 1.0 mg of egg white lysozyme under identical assay conditions. The enzyme activity of the sample was calculated by comparison with standard (egg white lysozyme, 0.5 to 6 µg/mL). The amount of lysozyme secreted was expressed as lysozyme per trachea tissue weight (micrograms per gram).

Mucin Secretion With Only the Tracheal Lumen Exposed to Hyperosmolar Solution
To evaluate whether hyperosmolar solution stimulates tracheal secretion via the luminal side in this model, whole trachea were used. Saline solution, 1.69 g/dL, or KHS was added into the inside of the trachea, and both ends of the trachea were tied. These tied tracheae (n = 2 each) were incubated for 20 min at 38°C with 1.69 g/dL (597 mOsm) of saline solution or KHS (288 mOsm/L) as follows: (1) inside saline solution-outside KHS, (2) outside saline solution–inside KHS, or (3) control inside and outside KHS. The inside solutions were collected and diluted with 10 mL of KHS for mucin analysis.

Evaluation of Secretory Pathways Using Inhibitors of Mucin Secretion
We also evaluated the secretory response to hyperosmolar solution along with inhibitors. After 2 h of resting in KHS, ferret tracheal segments were incubated for 20 min with each inhibitor in KHS or only in KHS as a control (period 1), and then incubated for another 20 min in hyperosmolar solution with each inhibitor in KHS, or with only KHS (negative control) or with hyperosmolar solution only (period 2). Atropine, 104 mol/L, was used as a muscarinic antagonist at a concentration shown to inhibit methacholine-induced tracheal secretion (104 mol/L of atropine against 105 mol/L of methacholine).11 We used nordihydroguaiaretic acid (NDGA), 105 mol/L, as an inhibitor of the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism.17 18 NDGA is also a free-radical scavenger. We also used protease inhibitors containing phenylmethylsulfonyl fluoride as an inhibitor of serine protease, leupeptin as a nonspecific protease inhibitor, and pepstatin A as an acid protease inhibitor. All agents were purchased from Sigma Chemical (St. Louis, MO).

Morphologic Evaluation of the Tracheal Epithelium
Tracheal segments (n = 4 each) were taken before and after immersion in 3.69 g/dL of saline solution (1,192 mOsm), 10.69 g/dL of saline solution (3,612 mOsm), or in KHS (288 mOsm). Tissue segments were immersed in 10% formalin and processed for light (wide-field) microscopy. Paraffin-embedded tissue was cut to 4-µm thickness, and slides were stained with hematoxylin and eosin. These were examined by a trained investigator masked as to treatment group (J.S.K.). He randomly selected eight fields for evaluation of epithelial damage.

Statistical Analysis
Statistical analysis of data were performed using the StatView 5 statistics package (SAS Institute; Cary, NC). Data were analyzed by analysis of variance Scheffe F test or Mann-Whitney U test to assess secretory response and the effect of inhibitors. Studies were powered to detect a 20% difference in measured secretion with a power of 0.85. Conventionally, p values < 0.05 were considered statistically significant. All data are presented as means ± SEM.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mucin and Lysozyme Secretion
There was a dose-related increase in mucin secretion compared to KHS (0.69 g/dL of saline solution [288 mOsm/L], 3,572 ± 291 ng/g secreted mucin, n = 16) after incubation with 1.69 g/dL of saline solution (597 mOsm/L) [6,264 ± 520 ng/g, 77% increase, n = 7; p < 0.01 for SI]; 3.69 g/dL of saline solution (1,192 mOsm) [7,227 ± 516 ng/g, 106% increase, n = 16; p < 0.0001]; 5.69 g/dL of saline solution (1,823 mOsm) [7,440 ± 490 ng/g, 116% increase, n = 11; p < 0.0001]; and 10.69 g/dL of saline solution (3,612 mOsm) [7,695 ± 389 ng/g, 130% increase, n = 10; p < 0.0001]. Mucin secretion was also significantly increased after exposure to 15 g/dL of mannitol (1,040 mOsm) [8,415 ± 606 ng/g, 135% increase, n = 4; p < 0.005; Fig 1 ].



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  		Figure 1. The effect of saline or mannitol solutions on mucin secretion. After 2 h of resting in KHS, ferret tracheal segments were incubated for 20 min with KHS (288 mOsm/L) [period 1], and then incubated for another 20 min in a hyperosmolar or KHS solution (period 2). An SI expressing the relative increase in mucin was calculated for each tracheal segment as the concentration of period 2 divided by that of period 1. The increased mucin secretion is presented as percentage increase compared to KHS. Data are presented as mean ± SEM. There was a dose-related increase in mucin secretion after incubation with 1.69 g/dL (597 mOsm/L) [n = 7, p < 0.01], 3.69 g/dL (1,192 mOsm/L) [n = 16, p < 0.0001], 5.69 g/dL (1,823 mOsm/L) [n = 11, p < 0.0001], and 10.69 g/dL (3,612 mOsm/L) [n = 10, p < 0.0001] of saline solution, and with 15 g/dL (1,040 mOsm/L) of mannitol (n = 4, p < 0.005) when compared to KHS control.

 
There was 47% increase in lysozyme secretion with exposure to 3.69 g/dL of saline solution and a 54% increase with exposure to 10.69 g/dL of saline solution (each n = 5, p < 0.05) compared with exposure to KHS (Fig 2 ). The amounts of lysozyme secretion in tracheae exposed to KHS, 3.69 g/dL of saline solution, and 10.69 g/dL of saline solution were 3.19 ± 0.38 µg/g, 4.58 ± 0.37 µg/g, and 4.68 ± 0.27 µg/g, respectively. The calculated ratio of mucin to lysozyme secretion was 2.0 in isotonic solution (KHS) as well as in 3% saline solution and 10% saline solution, suggesting that osmolar challenge induces a similar degree of secretion from mucous and serous glands.



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  		Figure 2. The effect of 3.69 g/dL and 10.69 g/dL saline solutions on lysozyme secretion. There was a 47% and 54% increase in lysozyme secretion after exposure to 3% and 10% saline solution, respectively (n = 5). Data are mean ± SEM for 3.69 g/dL and 10.69 g/dL saline solutions (p < 0.05), as compared to KHS-treated group (analysis of variance Scheffe F test).

 
The Kinetics of 3.69 g/dL Saline Solution-Induced Mucin Secretion
Tracheal segments were immersed in 3.69 g/dL of saline solution (1,192 mOsm) or KHS, and the buffers were collected at 5, 10, 15, and 30 min after incubation. Mucin secretion was induced immediately, and rapidly reached a maximum (Fig 3 ).



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  		Figure 3. Kinetics of mucin secretion after exposure to 3.69 g/dL of saline solution. The segments were immersed 3.69 g/dL (1,192 mOsm/L) in saline solution or KHS (288 mOsm/L) [each group, n = 16], and the solution was collected for mucin analysis 5, 10, 15, and 30 min after incubation. Data are mean ± SEM. *p < 0.0001, compared to KHS-treated group (Mann Whitney U test).

 
Hyperosmolar Solution Stimulates Mucin Secretion Only via the Luminal Side
Mucin secretion was stimulated only when the luminal side of the intact trachea was incubated with 1.69 g/dL of saline solution (597 mOsm/L), and the degree of stimulation was the same as when both the outside and the luminal side of the trachea were exposed (Fig 4 ).



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  		Figure 4. Hyperosmolar solution-stimulated mucin secretion via the epithelial surface. Saline solution (1.69 g/dL, 597 mOsm/L) or KHS (0.69 g/dL NaCl, 288 mOsm/L) was added into the inside of a whole trachea, and both ends of the trachea were tied. These tied tracheae (n = 2 each) were incubated for 20 min at 38°C with 1.69 g/dL of saline solution or KHS, as follows: (1) inside saline-outside KHS, (2) outside saline–inside KHS, or (3) control, inside and outside KHS. After a 20-min exposure, the inside solutions were collected and mucin was measured. Group 2 was significantly different (p < 0.05) from group 1 and group 3.

 
Evaluation of Secretory Pathways
Hyperosmolar saline solution-induced mucin and lysozyme secretion was not significantly inhibited by 104 mol/L of atropine, proteinase inhibitors, or 105 mol/L of NDGA (Fig 5 ).



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  		Figure 5. Effect of inhibitors of mucin secretion on osmolar-induced secretion. Tracheal segments were incubated for 20 min with each individual inhibitor in KHS or in only KHS (period 1), and then incubated for another 20 min in 3.69 g/dL (1,193 mOsm/L) in saline solution alone (positive control), with saline solution and each inhibitor in KHS, or with only KHS (negative control) [period 2]. Mucin secretion per gram of tissue during period 2 is shown. Inhibitors used were atropine at 104 mol/L, NDGA at 105 as an inhibitor of arachidonic acid metabolites, and a protease inhibitor cocktail (PIC) containing phenylmethylsulfonyl fluoride as an inhibitor of serine protease, leupeptin as a nonspecific protease inhibitor, and pepstatin A as an acid protease inhibitor. Error bars = SEM; p = not significant.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hyperosmolar inhalation produces a dose-dependent increase in mucin secretion in the cat trachea7 and in the human nose.19 Our results are consistent with these studies, showing that hyperosmolar saline solution or mannitol induced mucin secretion in a dose-related fashion. We demonstrated that hyperosmolar solutions stimulated both mucin and lysozyme secretion, suggesting that either there is co-regulation of secretion or that both mucous and serous cells respond in the same way to the hyperosmolar agents. This is supported by the observation that the rate of mucin secretion was approximately two times that of lysozyme secretion without stimulation and under all conditions of stimulation.

Mannitol appeared to induce mucin secretion to a greater extent than saline solution with similar osmolarity, although this was not statistically significant. This is consistent with a report that increased mucin secretion in the cat trachea was greater after incubation with sucrose solution than with hypertonic saline solution at a similar osmolarity.7 The tracheal epithelium is more permeable to chloride ion than to mannitol, leading to a more rapid equilibration of osmolarity about the epithelium and across the air side of the cell; therefore, the local gradient in osmolarity around the epithelium is probably less with saline solution exposure than with mannitol.

To understand the mechanism of hyperosmolar solution-induced airway secretion, we examined the effects of secretagogue inhibitors. In the rat trachea, hyperosmolar solutions can induce plasma exudation through neurogenic inflammation.20 Cholinergic antagonists and tachykinins can both cause vasodilatation, plasma exudation, and mucus secretion.21 22 23 Hyperosmolarity can also induce leukotriene and prostaglandin production,24 25 and these arachidonic acid metabolites can directly stimulate mucin secretion; however, neither atropine nor NDGA decreased saline solution-induced mucin secretion in this study.

Hyperosmolar solutions probably induce secretion through a direct action on secretory cells or by release of mediators that secondarily trigger receptor-mediated secretion. Because all central neural connections are severed in the excised ferret trachea, this secretagogue effect could not be due to central reflexes. Although secretion might have been induced by epithelial damage after osmotic challenge, there was no gross morphologic damage to the airway epithelium at the level of light microscopy when comparing KHS to saline solution-treated groups at 3.69 g/dL or 10.69 g/dL. This is consistent with reports that there was no visible injury to the epithelial or endothelial airway barriers after inhalation of 3% saline solution,26 and consistent with the observation that the proteinase inhibitors studied did not affect saline solution-induced mucin secretion.

Clinical studies show that hypertonic saline solution or mannitol inhalation increases MCC in both healthy and asthmatic subjects, apparently peaking in the first 10 to 15 min with little, if any, residual effect after 1 h.4 5 6 27 28 Although inhaled hyperosmolar agents can increase ciliary beat frequency (CBF), it is unlikely that this produces a clinically important effect. Hyperosmolar saline solution has been reported to increase, decrease, or have no effect on CBF. A very high concentration saline aerosol (3.4 mol/L, 19.9 g/dL) increased CBF in the canine airway.29 An increase in CBF was also observed with a 200 to 400 mOsm (0.58 to 1.17 g/dL) of saline solution in the mucus-depleted bovine trachea, but this was not dose dependent even though the MCC increased in a saline solution dose-dependent manner.8 A change of airway surface fluid osmolarity was reported to have no affect on CBF in the human airway.30 Finally, it has been reported that hypertonic salt solution decreases the CBF of chicken embryo cilia.31 This suggests that the increase in mucus transport after saline solution inhalation is probably due to other factors such as changes in mucus properties8 or acute secretion of preformed mucus in response to the hyperosmolarity.

We chose concentrations of hyperosmolar solutions based on concentrations used in clinical studies.3 4 5 6 15 We recognize that immersion of a tracheal segment into a solution is different from inhaling the same hyperosmolar solution, as immersion allows equilibration between the intracellular and extracellular spaces; however, we have demonstrated that mucin secretion was stimulated only when the luminal side of the trachea was incubated with hyperosmolar solution. As well, kinetic studies confirm a very rapid onset of action and a short time to maximal stimulation, suggesting a direct stimulation that is independent of tissue osmotic equilibration. We speculate that the increased mucus output and mucus clearance in response to hyperosmolarity may be a protective response to prevent airway fluid loss.

With hyperosmolar challenge, mucin and lysozyme appear to be secreted together rather than independently, and we hypothesize that this is due to osmotic changes and therefore not specific for the secretion of mucous or serous secretions. The ratio of mucin to lysozyme secretion remained consistently at 2:1 in iso-osmolar (KHS) conditions as well as in 3% and 10% saline solutions. It is likely that serous secretions are important for flushing mucus from submucosal glands. In fact, this is one of the hypotheses regarding mucus gland obstruction in CF. The CF transmembrane regulator protein is most highly expressed in the serous cells of submucosal glands, and perhaps serous cell secretion is impaired in the CF airway, preventing effective secretion of mucins from submucosal glands.

Extrapolating these results to patients with airway disease should be done cautiously. The study reported here was performed in healthy ferrets, and the secretagogue effect could be different when there is airway disease. Nevertheless, the data presented in this article suggest that the therapeutic use of hyperosmolar aerosols may be of benefit when used for patients with impaired mucus clearance.


   Footnotes
 
Abbreviations: CBF = ciliary beat frequency; CF = cystic fibrosis; DBA = Dolichos biflorus agglutinin; KHS = Krebs-Henseleit solution; MCC = mucociliary clearance; NDGA = nordihydroguaiaretic acid; PBS = phosphate-buffered saline solution; SI = secretory index

Dr. Kim was supported by a grant from the Cystic Fibrosis Foundation. Dr. Okamoto was an Abbott Research Fellow.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (e-mail: permissions{at}chestnet.org).

Received for publication November 8, 2001. Accepted for publication January 23, 2003.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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